α-Conotoxin BuIA[T5A;P6O]: a novel ligand that discriminates between α6β4 and α6β2 nicotinic acetylcholine receptors and blocks nicotine-stimulated norepinephrine release

alpha 6* (asterisk indicates the presence of additional subunits) nicotinic acetylcholine receptors (nAChRs) are broadly implicated in catecholamine-dependent disorders that involve attention, motor movement, and nicotine self-administration. Different molecular forms of alpha 6 nAChRs mediate catecholamine release, but receptor differentiation is greatly hampered by a paucity of subtype selective ligands. alpha-Conotoxins are nAChR-targeted peptides used by Conus species to incapacitate prey. We hypothesized that distinct conotoxin-binding kinetics could be exploited to develop a series of selective probes to enable study of native receptor subtypes. Proline6 of alpha-conotoxin BuIA was found to be critical for nAChR selectivity; substitution of proline6 with 4-hydroyx-proline increased the IC(50) by 2800-fold at alpha 6/alpha 3 beta 2 beta 3 but only by 6-fold at alpha 6/alpha 3 beta 4 nAChRs (to 1300 and 12 nM, respectively). We used conotoxin probes together with subunit-null mice to interrogate nAChR subtypes that modulate hippocampal norepinephrine release. Release was abolished in alpha 6-null mutant mice. alpha-Conotoxin BuIA[T5A;P6O] partially blocked norepinephrine release in wild-type controls but failed to block release in beta 4(-/-) mice. In contrast, BuIA[T5A;P6O] failed to block dopamine release in the wild-type striatum known to contain alpha 6 beta 2* nAChRs. BuIA[T5A;P6O] is a novel ligand for distinguishing between closely related alpha 6* nAChRs; alpha 6 beta 4* nAChRs modulate norepinephrine release in hippocampus but not dopamine release in striatum.-Azam, L., Maskos, U., Changeux, J.-P., Dowell, C. D., Christensen, S., De Biasi, M., McIntosh, J. M. alpha-Conotoxin BuIA[T5A;P6O]: a novel ligand that discriminates between alpha 6 beta 4 and alpha 6 beta 2 nAChRs and blocks nicotine-stimulated norepinephrine release. FASEB J. 24, 5113-5123 (2010). www.fasebj.org